Compressible/expandable medical graft products, and methods for applying hemostasis

ABSTRACT

Described are expanded collagenous materials useful in hemostatic applications. Certain expanded collagenous materials can be prepared by treating a first collagenous material with an alkaline substance under conditions effective to expand the first collagenous material, recovering the expanded material, processing the expanded material to provide a foam, and chemically crosslinking the foam. Expanded materials can exhibit beneficial resilience, persistence and tissue generation characteristics when implanted, and can be used in the formation of highly porous medical implant bodies which can be compressed to fractions of their original volume and will thereafter substantially recover their original volume.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/074,441, filed Jun. 20, 2008, which is herebyincorporated herein in its entirety.

BACKGROUND

The present invention relates generally to improved extracellular matrixmaterials and, in certain aspects, to physically modified extracellularmatrix materials, medical devices prepared therefrom, and uses thereof.

Biomaterials have been used in a variety of medical applications,including joint repair and replacement; periodontal reconstruction;repair or replacement of injured, diseased or malformed bones andtissues; wound healing; and the treatment of burns and diabetic ulcers.Extracellular matrix (ECM) materials, including those derived fromsubmucosa and other tissues, are known tissue graft materials used inthese medical applications. See, e.g., U.S. Pat. Nos. 4,902,508,4,956,178, 5,281,422, 5,372,821, 5,554,389, 6,099,567, and 6,206,931.These materials are typically derived from a variety of biologicalsources including, for example, small intestine, stomach, the urinarybladder, skin, pericardium, dura mater, fascia, and the like.

Challenges remain in obtaining finished medical products derived fromharvested animal ECM materials that possess the necessary physicalproperties as well as biological performance properties when implantedin patients. Accordingly, there remain needs for improved andalternative biomaterials and medical products, as well as methods forpreparing and using them.

SUMMARY

In certain of its aspects, the present invention features uniquecollagenous matrix materials that exhibit beneficial properties relatingto implant persistence, tissue generation, compressivity and/orexpansivity, and/or other physical or biological properties, and tomethods for their preparation and use. Desirable matrix materialscomprise a denatured, expanded extracellular matrix material and possessan ability to persist when implanted and encourage the ingrowth ofvascular structures into the matrix.

In one embodiment, the invention provides a method for tissue biopsywith applied hemostasis, comprising removing a biopsy sample from alocation in a patient, and implanting a hemostatic biopsy plug at thelocation, wherein the biopsy plug includes a resilient foam body formedwith an extracellular matrix material that has been treated with analkaline medium sufficient to form an expanded extracellular matrixmaterial. In certain forms, such methods can include advancing a biopsydevice into tissue of a patient, cutting a biopsy sample from a locationin the tissue, removing the biopsy sample from the patient, andimplanting the hemostatic biopsy plug at the location.

Another embodiment of the invention provides a hemostatic tissue biopsyplug product comprising a resilient, hemostatic extracellular matrixfoam body sized for receipt at a tissue biopsy site, the foam plugformed with an extracellular matrix material that has been treated withan alkaline medium sufficient to form an expanded extracellular matrixmaterial, said foam plug compressible to a compressed condition having agreatest cross-sectional dimension not exceeding about 5 mm andexpandable to an expanded condition having a greatest cross-sectionaldimension of at least about 10 mm. Preferably, the plug is characterizedby the ability to expand from the compressed condition to the expandedcondition in less than 1 minute.

In another embodiment, the invention provides a product for applyinghemostasis to a biopsy site, comprising a cannulated device having alumen, the cannulated device advanceable to a tissue biopsy site. Theproduct further includes a hemostatic tissue biopsy plug as describedherein received in the lumen.

In another embodiment, the present invention provides a method forproviding hemostasis at a surgical site, comprising surgically treatingtissue at a site in a patient in such a manner as to cause bleeding atthe site, and applying a hemostatic extracellular matrix foam to thesite so as to cause hemostasis, the foam formed with an extracellularmatrix material that has been treated with an alkaline medium sufficientto form an expanded extracellular matrix material.

In a further embodiment, the invention provides a method for surgicalremoval of parenchymal tissue in a patient with applied hemostasis,comprising performing a partial nephrectomy or hepatectomy in a patientso as to cause bleeding in a kidney or liver, respectively, of thepatient, and applying a hemostatic extracellular matrix foam to thekidney or liver so as to cause hemostasis, the foam formed with anextracellular matrix material that has been treated with an alkalinemedium sufficient to form an expanded extracellular matrix material.

The invention also provides a method for preparing a compressiblemedical foam product comprising lyophilizing an extracellular matrixmaterial that has been expanded with an alkaline medium to form alyophilized extracellular matrix material foam, and contacting thelyophilized foam with a crosslinking agent to form a crosslinked foam.In certain embodiments, such methods can comprise the steps of washingthe expanded extracellular matrix material, charging the expandedextracellular matrix material to a mold, lyophilizing the expandedextracellular matrix material in the mold to form a lyophilizedextracellular matrix material foam, contacting the lyophilizedextracellular matrix material foam with a chemical crosslinking agent toform a crosslinked extracellular matrix material foam, and drying thecrosslinked extracellular matrix material foam.

Also provided is a compressible medical foam product comprising a dried,compressible foam body formed with an extracellular matrix solidmaterial that has been treated with an alkaline medium under conditionseffective to produce an expanded extracellular matrix collagen material,wherein the foam body has introduced chemical crosslinks sufficient toincrease the resiliency of the foam body.

Additional aspects as well as features and advantages of the inventionwill be apparent to those of ordinary skill in the art from thedescriptions herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a micrograph taken at 100× magnification of a surfaceview of an expanded small intestinal submucosa material.

FIG. 1B depicts a micrograph taken at 100× magnification of a surfaceview of a non-expanded small intestinal submucosa material.

FIG. 1C depicts a micrograph taken at 100× magnification of across-section view of an expanded small intestinal submucosa material.

FIG. 1D depicts a micrograph taken at 100× magnification of across-section view of a non-expanded small intestinal submucosamaterial.

FIG. 2A provides a perspective view of a device useful for delivering ahemostatic medical product as described herein.

FIG. 2B provides a perspective view of the device illustrated in FIG. 2Awhere the hemostatic product is partially deployed from the device.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of aspects of theinvention, reference will now be made to certain embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended. Any alterations and further modificationsin the illustrative materials, constructs or methods described herein,and further applications of the principles of the invention asillustrated herein, are contemplated as would normally occur to oneskilled in the art to which the invention pertains.

As disclosed above, certain aspects of the present invention involvehemostatic methods and materials useful in such methods, as well as foamor sponge form devices that are capable of compression to a compressedstate, and resilient expansion from that compressed state. Methods forpreparing and using such devices also constitute aspects of theinvention disclosed herein.

Inventive products and methods are disclosed herein by which modifiedphysical characteristics are imparted to extracellular matrix materialsby controlled contact with an alkaline substance. Notably, suchtreatment can be used to promote substantial expansion (i.e. greaterthan about 20% expansion) of the extracellular matrix material. Inaccordance with certain aspects of the invention, this expanded materialis processed into a variety of useful medical materials and devices. Incertain embodiments, it is preferred to expand the material to at leastabout 2, at least about 3, at least about 4, at least about 5, or evenat least about 6 times its original bulk volume. It will be apparent toone skilled in the art that the magnitude of expansion is related to theconcentration of the alkaline substance, the exposure time of thealkaline substance to the material, and temperature, among others. Thesefactors can be varied through routine experimentation to achieve amaterial having the desired level of expansion, given the disclosuresherein. Such expanded materials can be used for example in hemostaticmethods and in the preparation of novel materials and devices forms asdiscussed further herein.

A collagen fibril is comprised of a quarter-staggered array oftropocollagen molecules. The tropocollagen molecules themselves areformed from three polypeptide chains linked together by covalentintramolecular bonds and hydrogen bonds to form a triple helix.Additionally, covalent intermolecular bonds are formed between differenttropocollagen molecules within the collagen fibril. Frequently, multiplecollagen fibrils assemble with one another to form collagen fibers. Itis believed that the addition of an alkaline substance to the materialas described herein will not significantly disrupt the intramolecularand intermolecular bonds, but will denature the material to an extentthat provides to the material a processed thickness that is at leasttwice the naturally-occurring thickness. In this regard, denaturation ofthe collagenous material to the extent described above allows for theproduction of a novel collagenous matrix material. The collagenousmatrix material comprises a sterile, processed collagenous matrixmaterial derived from a collagenous animal tissue layer, the collagenousanimal tissue layer has a naturally-occurring thickness and includes anetwork of collagen fibrils having naturally-occurring intramolecularcross links and naturally-occurring intermolecular cross links. Thenaturally-occurring intramolecular cross links and naturally-occurringintermolecular cross links have been retained in the sterile, processedcollagenous matrix material sufficiently to maintain the sterile,collagenous matrix material as an intact collagenous sheet material, andthe collagen fibrils as they occur in the intact collagenous sheetmaterial are denatured to an extent that provides to the intactcollagenous sheet material a processed thickness that is substantiallygreater (i.e. at least about 20% greater) than, and preferably at leasttwice the naturally-occurring thickness of, the collagenous animaltissue layer.

Turning now to the figures, FIGS. 1A-D depict surface andcross-sectional views of both an expanded and a non-expandedextracellular matrix material sheet (porcine small intestine submucosa)wherein collagen has been stained such that its content and structurecan be visualized. The four micrographs shown are as follows: (1A) thesurface of the expanded ECM sheet material, (1B) the surface of anon-expanded ECM sheet material, (1C) a cross section of the expandedECM sheet material, and (1D) a cross section of the non-expanded ECMsheet material. As shown in the micrographs, the surface and crosssection views of the non-expanded material exhibit a tightly boundcollagenous network whereas the same views of an expanded materialexhibit a denatured, but still intact, collagenous network which hasresulted in the expansion of the material.

In addition to causing expansion of a remodelable collagenous material,the application of an alkaline substance can alter the collagen packingcharacteristics of the material as illustrated in FIGS. 1A-D. Alteringsuch characteristics of the material can be caused, at least in part, bythe disruption of the tightly bound collagenous network. A non-expandedremodelable collagenous material having a tightly bound collagenousnetwork typically has a continuous surface that is substantially uniformeven when viewed under magnification, e.g. 100× magnification as shownin the Figures. Conversely, an expanded remodelable collagenous materialtypically has a surface that is quite different in that the surface istypically not continuous but rather presents collagen strands or bundlesin many regions that are separated by substantial gaps in materialbetween the strands or bundles. Consequently, an expanded remodelablecollagenous material typically appears more porous than a non-expandedremodelable collagenous material. Moreover, the expanded remodelablecollagenous material can be demonstrated as having increased porosity,e.g. by measuring its permeability to water or other fluid passage. Themore foamy and porous structure of an expanded remodelable collagenousmaterial can allow the material to be easily cast into a variety of foamshapes for use in the preparation of medical materials and devices. Itcan further allow for the compression and subsequent expansion of thematerial, which is useful, for example, when the material needs to beloaded into a deployment device for delivery into a patient. Oncedelivered, the material can expand to its original form.

As noted above, a non-expanded remodelable collagenous ECM material cantypically comprise a variety of bioactive components including, forexample, growth factors, glycoproteins, glycosaminoglycans,proteoglycans, nucleic acids, and lipids. Treating the material with analkaline substance under conditions as described herein cansignificantly reduce, if not completely eliminate, these bioactivecomponents from the material. Indeed, the treatment of the remodelablecollagenous material with an alkaline substance can result in aremodelable collagenous material which is substantially devoid of growthfactors, glycoproteins, glycosaminoglycans, proteoglycans, nucleicacids, and lipids. Accordingly, the treatment of a remodelablecollagenous material with an alkaline substance as described herein cancause the material to expand to at least about twice its originalvolume, can alter the surface and/or porosity characteristics of thematerial, and can deplete the material of certain bioactive components.In some embodiments, this is accomplished while maintaining the materialas an intact collagenous sheet, wherein the sheet can be furtherprocessed into any of a variety of medical materials and/or devices.Further, the remodelable collagenous material, such as an ECM sheet, canbe treated with the alkaline medium so as to expand it as describedherein, while the material retains an amount of a growth factor such asFGF-2, or another bioactive component such as fibronectin and/orheparin, that is/are native to the source tissue for the ECM or othercollagenous material.

In certain embodiments, selected bioactive components that werepreviously removed from the remodelable collagenous material can bereturned to the material. For example, the present invention provides anexpanded remodelable collagenous material, which is substantially devoidof nucleic acids and lipids, but which has been replenished with one ormore growth factors, glycoproteins, glycosaminoglycans, or proteoglycansor combinations thereof. These bioactive components can be returned tothe material by any suitable method. For instance, in certain forms, atissue extract containing these components can be prepared and appliedto an expanded remodelable collagenous material. In one embodiment, theexpanded remodelable collagenous material form is incubated in a tissueextract for a sufficient time to allow the bioactive componentscontained therein to associate with the expanded remodelable collagenousmaterial. The tissue extract may, for example, be obtained fromnon-expanded remodelable collagenous tissue of the same type used toprepare the expanded material. Other means for returning or providingbioactive components to an expanded remodelable collagenous materialinclude spraying, impregnating, dipping, etc. as known in the art. Byway of example, an expanded remodelable collagenous material may bemodified by the addition of one or more growth factors such as basicfibroblast growth factor (FGF-2), transforming growth factor beta (TGFbeta), epidermal growth factor (EGF), platelet derived growth factor(PDGF), and/or cartilage derived growth factor (CDGF). As well, anexpanded remodelable collagenous material may be replenished with otherbiological components such as heparin, heparin sulfate, hyaluronic acid,fibronectin and the like. Thus, generally speaking, an expandedremodelable collagenous material may include a bioactive component thatinduces, directly or indirectly, a cellular response such as a change incell morphology, proliferation, growth, protein or gene expression.

The preparation of submucosa extracts is described in, for example, U.S.Pat. No. 6,375,989. Briefly, a submucosa extract can be prepared by theaddition of an extraction excipient, such as urea, guanidine, sodiumchloride, magnesium chloride, or a surfactant, to a submucosa tissue toisolate bioactive components from the tissue. The bioactive componentsare then separated from the extraction excipient. In one preferredembodiment, a submucosa extract is prepared by mixing submucosa tissuewith a phosphate buffered solution, such as phosphate buffered saline(PBS). This mixture is processed into a slurry as buffer circulation andphysical pressure are applied. The bioactive components present in thetissue are drawn into solution and subsequently isolated from theslurry. The bioactive submucosa extract is then formed by separating theextracted bioactive components in the solution from the slurry usingart-recognized procedures such as dialysis and/or chromatographictechniques. Preferably, the extraction solution is dialyzed to reduce orremove the concentration of extraction excipients to provide a solutionof the extracted bioactive components. Any source of submucosa tissuecan be used to prepare a submucosa extract. Moreover, similar extractiontechniques can be applied to other remodelable ECM materials to providebiologically active extracts for use in the invention.

The nature and quantity of the bioactive components contained in thesubmucosa or other extracellular matrix (ECM) extract is dependent onthe nature and composition of the extraction excipients used for theextraction solution. Thus, for example, 2 M urea in a pH 7.4 bufferprovides an extracted submucosa fraction enriched for basic fibroblastgrowth factor and fibronectin, while 4 M guanidine in the same bufferprovides an extracted submucosa fraction enriched for a compoundexhibiting an activity profile for TGF-beta. Use of other extractionexcipients provides bioactive extracts comprising proteoglycans,glycoproteins and glycosaminoglycans such as heparin, heparin sulfate,hyaluronic acid, chondroitin sulfate A and chondroitin sulfate B.

In addition or as an alternative to the inclusion of native bioactivecomponents, such as those provided in a submucosa or other ECM extract,non-native bioactive components including those synthetically producedby recombinant technology or other methods, may be incorporated into theexpanded remodelable collagenous material. These non-native bioactivecomponents may be naturally-derived or recombinantly produced proteinsthat correspond to those natively occurring in the ECM tissue, butperhaps of a different species (e.g. human proteins applied tocollagenous ECMs from other animals, such as pigs). The non-nativebioactive components may also be drug substances. Illustrative drugsubstances that may be incorporated into and/or onto the expandedremodelable collagenous materials used in the invention include, forexample, antibiotics, thrombus-promoting substances such as bloodclotting factors, e.g. thrombin, fibrinogen, and the like. As with thebioactive components previously described, these substances may beapplied to the expanded remodelable collagenous material as apremanufactured step, immediately prior to the procedure (e.g. bysoaking the material in a solution containing a suitable antibiotic suchas cefazolin), or during or after engraftment of the material in thepatient.

The expanded remodelable collagenous material may also exhibit anangiogenic character and thus be effective to induce angiogenesis in ahost engrafted with the material. Angiogenic growth factors are wellknown in the art and include, for example, angiogenin, angiopoietin-1,Del-1, fibroblast growth factors (both acidic and basic), follistatin,granulocyte colony-stimulating factor, hepatocyte growth factor,interleukin-8 (IL-8), leptin, midkine, placental growth factor, plateletderived growth factor (PDGF), pleiotrophin, proliferin, transforminggrowth factors (both alpha and beta), tumor necrosis growth factor, andvascular endothelial growth factor (VEGF). Angiogenesis is the processthrough which the body makes new blood vessels to generate increasedblood supply to tissues. Thus, angiogenic materials, when contacted withhost tissues, promote or encourage the formation of new blood vessels.Methods for measuring in vivo angiogenesis in response to biomaterialimplantation have recently been developed. For example, one such methoduses a subcutaneous implant model to determine the angiogenic characterof a material. See, C. Heeschen et al., Nature Medicine 7 (2001), No. 7,833-839. When combined with a fluorescence microangiography technique,this model can provide both quantitative and qualitative measures ofangiogenesis into biomaterials. C. Johnson et al., Circulation Research94 (2004), No. 2, 262-268.

Expanded remodelable collagenous materials, as well as tissue extractsas described herein, are prepared, for example, from collagenousmaterials isolated from a suitable tissue source from a warm-bloodedvertebrate, and especially a mammal. Such isolated collagenous materialcan be processed so as to have remodelable properties and promotecellular invasion and ingrowth. Suitable remodelable materials can beprovided by collagenous extracellular matrix (ECM) materials possessingbiotropic properties.

Suitable bioremodelable materials can be provided by collagenousextracellular matrix materials (ECMs) possessing biotropic properties,including in certain forms angiogenic collagenous extracellular matrixmaterials. For example, suitable collagenous materials include ECMs suchas submucosa, renal capsule membrane, dermal collagen, dura mater,pericardium, fascia lata, serosa, peritoneum or basement membranelayers, including liver basement membrane. These and other similaranimal-derived tissue layers can be expanded and processed as describedherein. Suitable submucosa materials for these purposes include, forinstance, intestinal submucosa, including small intestinal submucosa,stomach submucosa, urinary bladder submucosa, and uterine submucosa.

Submucosa or other ECM tissue used in the invention is preferably highlypurified, for example, as described in U.S. Pat. No. 6,206,931 to Cooket al. Thus, preferred ECM material will exhibit an endotoxin level ofless than about 12 endotoxin units (EU) per gram, more preferably lessthan about 5 EU per gram, and most preferably less than about 1 EU pergram. As additional preferences, the submucosa or other ECM material mayhave a bioburden of less than about 1 colony forming units (CFU) pergram, more preferably less than about 0.5 CFU per gram. Fungus levelsare desirably similarly low, for example less than about 1 CFU per gram,more preferably less than about 0.5 CFU per gram. Nucleic acid levelsare preferably less than about 5 μg/mg, more preferably less than about2 μg/mg, and virus levels are preferably less than about 50 plaqueforming units (PFU) per gram, more preferably less than about 5 PFU pergram. These and additional properties of submucosa or other ECM tissuetaught in U.S. Pat. No. 6,206,931 may be characteristic of the submucosatissue used in the present invention.

In order to prepare an expanded remodelable collagenous material, thematerial is preferably treated with a disinfecting agent so as toproduce a disinfected, expanded remodelable collagenous material.Treatment with a disinfecting agent can be done either prior to or afterisolation of the remodelable collagenous material from the tissue sourceor can be done either prior to or after expansion. In one preferredembodiment, the tissue source material is rinsed with a solvent, such aswater, and is subsequently treated with a disinfecting agent prior todelamination. It has been found that by following thispost-disinfection-stripping procedure, it is easier to separate theremodelable collagenous material from the attached tissues as comparedto stripping the remodelable collagenous material prior to disinfection.Additionally, it has been discovered that the resultant remodelablecollagenous material in its most preferred form exhibits superiorhistology, in that there is less attached tissue and debris on thesurface compared to a remodelable collagenous material obtained by firstdelaminating the submucosa layer from its source and then disinfectingthe material. Moreover, a more uniform remodelable collagenous materialcan be obtained from this process, and a remodelable collagenousmaterial having the same or similar physical and biochemical propertiescan be obtained more consistently from each separate processing run.Importantly, a highly purified, substantially disinfected remodelablecollagenous material is obtained by this process. In this regard, oneembodiment of the invention provides a method for preparing an expandedremodelable collagenous material. The method comprises providing atissue source including a remodelable collagenous material, disinfectingthe tissue source, isolating the remodelable collagenous material fromthe tissue source, and contacting the disinfected remodelablecollagenous material with an alkaline substance under conditionseffective to expand the remodelable collagenous material to at leastabout two times its original volume, thereby forming the expandedremodelable collagenous material. Upon formation of the expandedremodelable collagenous material, the material can be further processedinto medical materials and/or devices, or can be stored, e.g. in highpurity water at 4° C., for later use.

Preferred disinfecting agents are desirably oxidizing agents such asperoxy compounds, preferably organic peroxy compounds, and morepreferably peracids. As to peracid compounds that can be used, theseinclude peracetic acid, perpropioic acid, or perbenzoic acid. Peraceticacid is the most preferred disinfecting agent for purposes of thepresent invention. Such disinfecting agents are desirably used in aliquid medium, preferably a solution, having a pH of about 1.5 to about10, more preferably a pH of about 2 to about 6, and most preferably a pHof about 2 to about 4. In methods of the present invention, thedisinfecting agent will generally be used under conditions and for aperiod of time which provide the recovery of characteristic, purifiedsubmucosa materials as described herein, preferably exhibiting abioburden of essentially zero and/or essential freedom from pyrogens. Inthis regard, desirable processes of the invention involve immersing thetissue source or isolated remodelable collagenous material (e.g. bysubmersing or showering) in a liquid medium containing the disinfectingagent for a period of at least about 5 minutes, typically in the rangeof about 5 minutes to about 40 hours, and more typically in the range ofabout 0.5 hours to about 5 hours.

When used, peracetic acid is desirably diluted into about a 2% to about50% by volume of alcohol solution, preferably ethanol. The concentrationof the peracetic acid may range, for instance, from about 0.05% byvolume to about 1.0% by volume. Most preferably, the concentration ofthe peracetic acid is from about 0.1% to about 0.3% by volume. Whenhydrogen peroxide is used, the concentration can range from about 0.05%to about 30% by volume. More desirably the hydrogen peroxideconcentration is from about 1% to about 10% by volume, and mostpreferably from about 2% to about 5% by volume. The solution may or maynot be buffered to a pH from about 5 to about 9, with more preferredpH's being from about 6 to about 7.5. These concentrations of hydrogenperoxide can be diluted in water or in an aqueous solution of about 2%to about 50% by volume of alcohol, most preferably ethanol.

With respect to the alkaline substance used to prepare an expandedremodelable collagenous material, any suitable alkaline substancegenerally known in the art can be used. Suitable alkaline substances caninclude, for example, salts or other compounds that that providehydroxide ions in an aqueous medium. Preferably, the alkaline substancecomprises sodium hydroxide (NaOH). The concentration of the alkalinesubstance that is added to the material can be in the range of about 0.5to about 4 M. Preferably, the concentration of the alkaline substance isin the range of about 1 to about 3M. Additionally, the pH of thealkaline substance will typically range from about 8 to about 14. Inpreferred embodiments, the alkaline substance will have a pH of fromabout 10 to about 14, and most preferably of from about 12 to about 14.

In addition to concentration and pH, other factors such as temperatureand exposure time will contribute to the extent of expansion. In thisrespect, it is preferred that the exposure of the remodelablecollagenous material to the alkaline substance is performed at atemperature of about 4 to about 45° C. In preferred embodiments, theexposure is performed at a temperature of about 25 to about 37° C., with37° C. being most preferred. Moreover, the exposure time can range fromabout several minutes to about 5 hours or more. In preferredembodiments, the exposure time is about 1 to about 2 hours. In aparticularly preferred embodiment, the remodelable collagenous materialis exposed to a 3 M solution of NaOH having a pH of 14 at a temperatureof about 37° C. for about 1.5 to 2 hours. Such treatment results in theexpansion of a remodelable collagenous material to at least about twiceits original volume. As indicated above, these processing steps can bemodified to achieve the desired level of expansion.

In addition to an alkaline substance, a lipid removal agent can also beadded to a remodelable collagenous material either prior to, inconjunction with, or after the addition of the alkaline substance.Suitable lipid removal agents include, for example, solvents such asether and chloroform, or surfactants. Other suitable lipid removalagents will be apparent to those of ordinary skill in the art.Accordingly, the lipid removal agents listed herein serve only asexamples, and are therefore in no way limiting.

In preferred embodiments, the expanded remodelable collagenousmaterials, as well as tissue extracts containing bioactive componentsthat can optionally be added to an expanded remodelable collagenousmaterial, are sterilized using conventional sterilization techniquesincluding tanning with glutaraldehyde, formaldehyde tanning at acidicpH, ethylene oxide treatment, propylene oxide treatment, gas plasmasterilization, gamma radiation, and peracetic acid sterilization. Asterilization technique which does not significantly alter theremodelable properties of the expanded remodelable collagenous materialis preferably used. Moreover, in embodiments where the expandedremodelable collagenous material includes a native or non-nativebioactive component, the sterilization technique preferably does notsignificantly alter the bioactivity of the expanded remodelablecollagenous material. Preferred sterilization techniques includeexposing the extract to peracetic acid, low dose gamma irradiation (2.5mRad) and gas plasma sterilization.

The expanded remodelable collagenous materials of and for use in theinvention can be provided in any suitable form, including a flowableaqueous composition (e.g., a fluidized composition), a powder, a gel, asponge, one or more sheets, or a cast body. In one embodiment, theexpanded remodelable collagenous material is processed into a fluidizedcomposition, for instance using techniques as described in U.S. Pat. No.5,275,826. In this regard, solutions or suspensions of the expandedremodelable collagenous material can be prepared by comminuting and/ordigesting the material with a protease (e.g. trypsin or pepsin), for aperiod of time sufficient to solubilize the material and formsubstantially homogeneous solution. The expanded remodelable collagenousmaterial is desirably comminuted by, tearing, cutting, grinding,shearing (e.g. combined with a liquid and sheared in a blender), or thelike. The expanded remodelable collagenous material typically has aspongy and porous structure, so these techniques may not be needed tothe extent they would be needed to solubilize a non-expanded remodelablecollagenous material. Grinding the material in a frozen or freeze-driedstate is advantageous, although good results can be obtained as well bysubjecting a suspension of pieces of the material to treatment in a highspeed blender and dewatering, if necessary, by centrifuging anddecanting excess waste. The comminuted material can be dried, forexample freeze dried, to form a particulate. The particulate can be useditself to treat a patient, e.g., for trauma wounds, or can be hydrated,that is, combined with water or buffered saline and optionally otherpharmaceutically acceptable excipients, to form a fluidized, expandedremodelable collagenous material, e.g. having a viscosity of about 2 toabout 300,000 cps at 25° C. The higher viscosity graft compositions canhave a gel or paste consistency.

In one embodiment of the invention, a particulate remodelablecollagenous material formed separately from the expanded remodelablecollagenous material can be combined with a fluidized, expandedremodelable collagenous material. Such particulate remodelablecollagenous materials can be prepared by cutting, tearing, grinding,shearing or otherwise comminuting a remodelable collagenous sourcematerial. Such a material can be an expanded material or a non-expandedmaterial. As well, the expanded or non-expanded particulate can includeone or more additives to promote hemostasis. Suitable such additivesinclude, as examples, calcium alginate or zeolite. Such additives caninclude adhesive properties that allow the particulate to adhere to adesired location (e.g., tissue surface) after implantation. For example,a particulate ECM material having an average particle size of about 50microns to about 500 microns may be included in the fluidized, expandedremodelable collagenous material, more preferably about 100 microns toabout 400 microns. The remodelable collagenous particulate can be addedin any suitable amount relative to the fluidized, expanded remodelablecollagenous material, with preferred remodelable collagenous particulateto fluidized, expanded remodelable collagenous material weight ratios(based on dry solids) being about 0.1:1 to about 200:1, more preferablyin the range of 1:1 to about 100:1. In these embodiments, theremodelable collagenous particulate material can be included at a sizeand in an amount that effectively retains an injectable character to thefluidized, expanded remodelable collagenous material, for example byinjection through a needle having a size in the range of 18 to 31 gauge(internal diameters of 0.047 inches to about 0.004 inches). Theinclusion of such remodelable collagenous particulates in the ultimatefluidized, expanded remodelable collagenous material can serve toprovide additional material that can function to provide bioactivity tothe composition (e.g. itself including growth factors or other bioactivecomponents as discussed herein), serve as scaffolding material fortissue ingrowth and/or promote expansion of a compressed remodelablecollagenous material. Further, such materials including both fluidizedexpanded remodelable collagenous material and remodelable collagenousparticulate material can optionally be processed to form dried productsincorporating both materials, e.g. dried foam products which can be usedfor hemostasis, occlusion or other purposes and which are optionallycrosslinked as disclosed herein.

It is contemplated that commercial products may constitute any of thethese forms of the fluidized, expanded remodelable collagenous material,e.g. (i) packaged, sterile powders which can be reconstituted in anaqueous medium to form a gel, or (ii) packaged, sterile aqueous gel orpaste compositions including expanded remodelable collagenous materialcomponents. In one embodiment of the invention, a medical kit includes apackaged, sterile, dried (e.g. lyophilized) expanded remodelablecollagenous material powder, and a separately packaged, sterile aqueousreconstituting medium. In use, the expanded remodelable collagenousmaterial powder can be reconstituted with the reconstituting medium toform a gel.

A fluidized composition prepared from an expanded remodelablecollagenous material as described herein can optionally be dried to forma sponge solid or foam material. Dry sponge or foam form materials ofthe invention prepared by drying expanded remodelable collagenousmaterial gels and can be used, for example, in wound healing, tissuereconstructive applications, occlusive applications, hemostaticapplications, in the culture of cells, and in a variety of additionalapplications including those disclosed elsewhere herein.

In embodiments of the invention where an expanded remodelablecollagenous ECM material is provided in sheet form, the material canhave a thickness in the range of about 0.2 mm to about 2 mm, morepreferably about 0.4 mm to about 1.5 mm, and most preferably about 0.5mm to about 1 mm. If necessary or desired, a multilaminate material canbe used. For example, a plurality of (i.e. two or more) layers of anexpanded remodelable collagenous ECM material can be bonded or otherwisecoupled together to form a multilaminate structure. Illustratively, two,three, four, five, six, seven, or eight or more layers of an expandedremodelable collagenous material can be bonded together to provide amultilaminate material. In certain embodiments, two to six expanded,submucosa-containing layers isolated from intestinal tissue of awarm-blooded vertebrate, particularly small intestinal tissue, arebonded together to provide a medical material. Porcine-derived smallintestinal tissue is preferred for this purpose. In alternativeembodiments, one or more sheets of a non-expanded collagenous material(e.g., submucosa) can be bonded or otherwise coupled to one or moresheets of an expanded remodelable collagenous material. Any number oflayers can be used for this purpose and can be arranged in any suitablefashion with any number of layers of a non-expanded remodelablecollagenous material bonded to any number of layers of an expandedremodelable collagenous material. The layers of collagenous tissue canbe bonded together in any suitable fashion, including dehydrothermalbonding under heated, non-heated or lyophilization conditions, usingadhesives as described herein, glues or other bonding agents,crosslinking with chemical agents or radiation (including UV radiation),or any combination of these with each other or other suitable methods.

A variety of dehydration-induced bonding methods can be used to fuseportions of multi-layered medical materials together. In one preferredembodiment, the multiple layers of material are compressed underdehydrating conditions. The term “dehydrating conditions” can includeany mechanical or environmental condition which promotes or induces theremoval of water from the multi-layered medical material. To promotedehydration of the compressed material, at least one of the two surfacescompressing the matrix structure can be water permeable. Dehydration ofthe material can optionally be further enhanced by applying blottingmaterial, heating the matrix structure or blowing air, or other inertgas, across the exterior of the compressing surfaces. One particularlyuseful method of dehydration bonding multi-layered medical materials islyophilization, e.g. freeze-drying or evaporative cooling conditions.

Another method of dehydration bonding comprises pulling a vacuum on theassembly while simultaneously pressing the assembly together. Thismethod is known as vacuum pressing. During vacuum pressing, dehydrationof the multi-layered medical materials in forced contact with oneanother effectively bonds the materials to one another, even in theabsence of other agents for achieving a bond, although such agents canbe used while also taking advantage at least in part of thedehydration-induced bonding. With sufficient compression anddehydration, the multi-layered medical materials can be caused to form agenerally unitary laminate structure.

It is advantageous in some aspects of the invention to perform dryingoperations under relatively mild temperature exposure conditions thatminimize deleterious effects upon the multi-layered medical materials ofthe invention, for example native collagen structures and potentiallybioactive substances present. Thus, drying operations conducted with noor substantially no duration of exposure to temperatures above humanbody temperature or slightly higher, say, no higher than about 38° C.,will preferably be used in some forms of the present invention. Theseinclude, for example, vacuum pressing operations at less than about 38°C., forced air drying at less than about 38° C., or either of theseprocesses with no active heating—at about room temperature (about 25°C.) or with cooling. Relatively low temperature conditions also, ofcourse, include lyophilization conditions. It will be understood thatthe above-described means for coupling two or more multi-layered medicalmaterials together to form a laminate can also apply for couplingtogether one or more layers of peritoneum and fascia when these layersare isolated independent from one another.

In addition to the above, the expanded remodelable collagenous materialof the present invention can be used to prepare a molded or shapedconstruct for example a sponge useful as an occluder device or biopsyplug. A method for preparing such device comprises providing an expandedremodelable collagenous material, comminuting the expanded material(e.g. to provide layer fragments of expanded remodelable collagenousmaterial), casting the comminuted expanded remodelable collagenousmaterial into a shape, and freezing and lyophilizing the cast, expandedremodelable collagenous material to form the construct. Freezing can bedone at a temperature of about −80° C. for about 1 to about 4 hours, andlyophilization can be performed for about 8 to about 48 hours.Typically, the material used to prepare the construct is an expandedremodelable collagenous material that can optionally be replenished withone or more bioactive components. The expanded remodelable collagenousmaterial can be cast into any shape desired, for example a size andshape to occlude a particular area in need of occlusion or to promotehemostasis. In certain preferred embodiments, a biopsy plug is formedand is used, for example, to fill a void in a tissue (e.g., organtissue) after surgery. When a sponge form construct is prepared, thelyophilized, expanded remodelable collagenous material can be compressedand loaded into a deployment device for delivery into a patient. Oncedelivered, the device can expand to occlude or provide hemostasis to thearea in which it was deployed. Suitable deployment devices will begenerally known to those of ordinary skill in the art and include, forexample, tubular devices such as delivery catheters and the like.

In certain embodiments, it may be desirable to include one or moreadditives into the expanded remodelable collagenous material to promotere-expansion of a compressed material. Any suitable additive can beused. Suitable additives include, for example, salts, such as sodiumchloride, sodium acetate, sodium bicarbonate, sodium citrate, calciumcarbonate, potassium acetate, potassium phosphate; hydrogel andwater-swelling polymers, such as alginate, polyhydroxethyl methacralate,polyhydroxypropyl methacrylate, polyvinyl alcohol, polyethylene glycol,carboxymethyl cellulose, polyvinyl pyrrolidone; proteins, such asgelatin and SIS particulate; acids and bases, such as acetic acid andascorbic acid; superabsorbing polymers and gelling agents, such aspolyacrylic acid, pectin, polygalacturonic acid, polyacrylicacid-co-acrylamide, polyisobutylene-co-maleic acid; monosaccharides,polysaccharides, and derivatives thereof, such as dextran, glucose,fructose, sucrose, sucrose ester, sucrose laurate, galactose, chitosan,poly-N-acetyl glucosamine, heparin, hyaluronan, and chrondroitinsulfate; as well as other potential additives, such as guanidine HCl,urea, hydroxyethyl cellulose, sodium cholate, sodium taurocholate, ionicdetergents (e.g., SDS), and non-ionic detergents (e.g., Triton). Inpreferred embodiments, the one or more additives includes abiocompatible salt such as sodium chloride, sodium acetate, or sodiumbicarbonate; polyethylene glycol (e.g. MW 6000), and/or SIS or other ECMparticulate.

Turning now to a brief overview of illustrative deployment devices andprocedures useful for delivering a dried, expanded material as describedherein, with general reference to FIGS. 2A and 2B, in certain aspects, adeployment system 20 can include a cannulated device 21, e.g. acatheter, sheath or other tube that can be used to house and deliver ahemostatic plug 24 as described herein. For example, the cannulateddevice 21 with the plug 24 housed therein can be maneuvered throughanother instrument 22. Instrument 22 can be any of a variety of surgicalinstruments, including for example a laproscope, endoscope (includinge.g. a nephroscope), or an outer vascular access or delivery sheath. Inembodiments wherein instrument 22 is an endoscopic instrument,instrument 22 will of course also typically include additional passagesor channels extending therethrough to provide, for example, fiber opticlight input, viewing function (e.g. with a telescope or camera), and thelike. In these embodiments, the irrigation or other working channel ofthe endoscope can be used to pass the cannulated device 21 for deliveryof the plug 24 to the target site.

A counterforce and/or pusher element 23 can be provided within the lumenof cannulated device 21 to facilitate delivery of the plug 24 out of theopen distal end of the cannulated device 21. The element 23 can beadvanced forward within device 21 to push plug 24 from device 21, orelement 23 can be held in position against the plug 24 while device 21is retracted to deliver plug 24 from device 21, or a combination ofthese two functions can be used. In alternative embodiments, othermethods may be used for delivering plug 24 from cannulated device 21,including as one example the use of a liquid under pressure to forceplug 24 from the open end of the cannulated device 21.

Illustratively, a plug 24 can be loaded within the cannulated device 21and can be deployed at a site for hemostasis (e.g. a biopsy site orwithin a needle tract through soft tissue resultant of a percutaneousaccess procedure) or otherwise within a bodily passage or void by usingone or more actuator members positioned external of the patient thatcontrol the relative position of the cannulated device 21 and thecounterforce/pusher element 23. The one or more actuator members caninclude manually operable triggers, rotatable knobs, or other elementsthat may be connected to device 21 and/or element 23 directly or throughcontrol wired, rods, or other suitable members known in the art. Incertain embodiments, system 20 can have one or more actuator member(s)that deploy the plug 24 in a stepwise fashion, such that a first manualoperation of the actuator(s) controllably delivers a predeterminedpercentage of the plug 24 from the open end of cannulated device 21leaving the plug 24 in a partially delivered state, an a second manualoperation of the actuator(s) delivers a further percentage of the plug24 from the open end of cannulated device. Such further percentage ispreferably the entire remainder of the plug 24, although systems mayalso be designed to deliver the plug fully upon multiple additionaloperations of the actuator(s). In certain embodiments, a first operationof the actuator member(s) deploys about 10% to about 70% of the lengthof plug 24 from the end of cannulated device 21, and a second operationof the actuator(s) delivers the remainder of the plug 24 from thecannulated device.

Deployment devices, including delivery sheaths, cannulated devices, andpushers, used in the invention can all be conventional marketed productsor modifications thereof. For example, sheaths can be formed from PTFE(e.g. Teflon), polyamide (e.g. Nylon) or polyurethane materials, or acombination of materials such as an assembly including an inner layer ofPTFE, a flat wire coil over the PTFE for kink resistance, and apolyamide (Nylon) outer layer to provide integrity to the overallstructure and a smooth surface (e.g. as in the Flexor sheath, Cook,Inc.). Pushers can be made from conventional materials such aspolyethylene, polyamide, polyurethane or vinyl, stainless steel, or anycombination of these materials. Catheters can be made from conventionalmaterials such as polyethylene, polyamide, PTFE, polyurethane, and othermaterials.

An expanded material as described herein can be compressed prior todelivery and can expand following deployment from the catheter until itcontacts inner surfaces of a bodily passage or void, a biopsy site orother surgically created void. With certain designs, this expansion andcontact will be sufficient to maintain the material at a particularlocation in the bodily passage or void following deployment, althoughsome inventive implants will incorporate one or more anchoring orsecurement adaptations (not shown) so as to mitigate undesirablemigration of the device from or within the passageway or void. In someinstances, parts of an expanded material can embed themselves in tissuessurrounding the void or passageway upon deployment. Any number ofanchoring adaptations, such as barbs, hooks, ribs, protuberances, and/orother suitable surface modifications can be incorporated into aninventive devices to anchor them during and/or after deployment.

Hemostatic products as described herein can be any suitable length andwill generally be of sufficient dimension to achieve hemostasis at adesired location e.g., a surgery site. In certain embodiments, a device,in implanted form, will have a length of at least about 0.4 cm, and inmany situations will have a length ranging from about 1 cm to about 30cm, more typically from about 2 cm to about 15 cm.

As noted above, one or more additives can provide a variety offunctions, including promoting expansion of the material once implantedinto a patient. For example, a sponge form expanded remodelablecollagenous material including one or more additives can be compressedand placed into a delivery device. Compression of the material allowsthe material to be more easily transferred to a patient. Upon delivery,the material can expand to at least about its original size prior tocompression. This is typically done with an occluder device or a biopsyplug where it is desirable for the material to have a smaller diameterprior to delivery and expand upon delivery. Such additives can beincluded in the remodelable collagenous material to expand the materialat a faster rate than would otherwise be achievable in the absence ofthe one or more additives. For example, one or more additives can beincluded with a compressed remodelable collagenous material so as topromote the re-expansion of the material back to its original sizewithin at least about 30 seconds, 45 seconds, 1 minute, 2 minutes, 3minutes, 4 minutes, or even about least about 5 minutes afterimplantation. As with the bioactive components previously described,these additives may be applied to the expanded remodelable collagenousmaterial as a premanufactured step, immediately prior to the procedure(e.g. by soaking the material in a solution containing a suitableantibiotic such as cefazolin), or during or after engraftment of thematerial in the patient.

As noted above, expanded remodelable collagenous materials can be formedinto a sponge construct for implantation into a patient. In certainembodiments, a sponge construct will be constructed such that thematerial does not fully expand until after delivery to a desired site(e.g., tissue defect). In these instances, an expanded remodelablecollagenous material can be encapsulated, either partially or wholly, soas to prevent the premature expansion of the material until it reachesits intended delivery site. For example, a dried sponge material asdescribed herein can be compressed and either partially or whollyencapsulated into a biodegradable capsule. In such embodiments, thecapsule can retain the material in a compressed state so as to preventthe premature expansion of the expanded remodelable collagenous materialduring delivery. This allows the material to be delivered to a desiredlocation before full expansion occurs. In a similar embodiment, anexpanded remodelable collagenous material in powder form can be providedin a biocompatible, biodegradable capsule for delivery. Such anembodiment retains the powder within the capsule so as to preventportions of the powder from being delivered or drifting to an unintendedlocation. Biocompatible materials suitable for use in forming abiodegradable capsule are generally known in the art and can include,for example, gelatin.

In certain embodiments, an expanded remodelable collagenous material, inany form, can be crosslinked. An expanded remodelable collagenousmaterial can be crosslinked either before or after it is formed into amedical device, or both. Increasing the amount (or number) ofcrosslinkages within the material or between two or more layers of thematerial can be used to enhance its strength. However, when aremodelable material is used, the introduction of crosslinkages withinthe material may also affect its resorbability or remodelability.Consequently, in certain embodiments, a remodelable collagenous materialwill substantially retain its native level of crosslinking, or theamount of added crosslinkages within the medical device will bejudiciously selected depending upon the desired treatment regime. Inmany cases, the material will exhibit remodelable properties such thatthe remodeling process occurs over the course of several days or severalweeks. In certain preferred embodiments, the remodeling process occurswithin a matter of about 5 days to about 12 weeks. With regard to asponge form construct, crosslinking of a compressed construct maypromote re-expansion of the construct after implantation into a patient.

With regard to compressible/expandable plugs, sponges or otherconstructs as described herein, expansion additives and/or crosslinkingcan be used to impart desirable compression/re-expansion properties. Inpreferred forms, the constructs are capable of volumetric compressionwhen dry at a ratio of at least 10:1 (i.e. the compressed form occupiesno more than 10% of its original, relaxed and unexpanded volume), morepreferably at a ratio of at least 20:1. At the same time, in preferredforms, the compressed constructs are capable of re-expansion tosubstantially their original volume (e.g. at least about 80% of theiroriginal volume, more preferably at least 90%, and most preferably atleast 95%) within about 30 seconds when delivered in their dry,compressed form into a volume of water.

For use in the present invention, introduced crosslinking of theexpanded remodelable collagenous material may be achieved byphoto-crosslinking techniques, or by the application of a crosslinkingagent, such as by chemical crosslinkers, or by protein crosslinkinginduced by dehydration or other means. Chemical crosslinkers that may beused include for example aldehydes such as glutaraldehydes, diimidessuch as carbodiimides, e.g.,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC),diisocyanates such as hexamethylene-diisocyanate, ribose or othersugars, acyl-azide, sulfo-N-hydroxysuccinamide, or polyepoxidecompounds, including for example polyglycidyl ethers such asethyleneglycol diglycidyl ether, available under the trade name DENACOLEX810 from Nagese Chemical Co., Osaka, Japan, and glycerol polyglycerolether available under the trade name DENACOL EX 313 also from NageseChemical Co. Typically, when used, polyglycerol ethers or otherpolyepoxide compounds will have from 2 to about 10 epoxide groups permolecule.

When a multi-layered laminate material is contemplated, the layers ofthe laminate can be additionally crosslinked to bond multiple layers ofa multi-layered medical material to one another. Cross-linking ofmulti-layered medical materials can also be catalyzed by exposing thematrix to UV radiation, by treating the collagen-based matrix withenzymes such as transglutaminase and lysyl oxidase, and byphotocrosslinking. Thus, additional crosslinking may be added toindividual layers prior to coupling to one another, during coupling toone another, and/or after coupling to one another.

The medical materials, constructs and devices of the invention can beprovided in sterile packaging suitable for medical materials anddevices. Sterilization may be achieved, for example, by irradiation,ethylene oxide gas, or any other suitable sterilization technique, andthe materials and other properties of the medical packaging will beselected accordingly.

In certain embodiments, the invention provides compressible medical foamproducts, and methods for their preparation. The medical foam productsinclude a dried, compressible foam body formed with an extracellularmatrix solid material that has been treated with an alkaline mediumunder conditions effective to produce an expanded extracellular matrixcollagen material. The foam body has introduced chemical crosslinkssufficient to increase the resiliency of the foam body. Absentcrosslinking, foam bodies produced from the expanded extracellularmatrix collagen material possess resiliency, but for certainapplications, including for example hemostatic plug applications, it hasbeen discovered that increased resiliency is desired. The introductionof collagen crosslinks, for example with chemical crosslinkers such asglutaraldehyde, carbodimides, or other chemical crosslinkers identifiedherein, has been found to significantly enhance the resiliency of thefoam plugs, while leaving the compressible to a small size for delivery.Increased resiliency in turn provides additional compression uponadjacent tissues when the foam plugs are inserted in a compressed stateand then allowed to expand in situ in a patient at a site at whichhemostasis is desired. In specific inventive applications, crosslinked,resilient foam plugs as disclosed herein can be utilized to providehemostasis at surgical sites, including biopsy sites. These biopsy orother surgical sites can be located within parenchymal organ tissues,such as those of a kidney, liver or spleen of a patient.

Thus, in certain forms of the invention, surgical methods are providedwhich include resecting tissue from a parenchymal organ such as a liveror kidney, and then implanting a crosslinked, resilient foam material asdescribed herein at the resection site so as to facilitate hemostasis.The resection can, as examples, occur as a part of a nephrectomy orhepatectomy, e.g. to removed cancerous or other diseased tissue, or as apart of a kidney or liver biopsy performed with a biopsy needle. In thecase of minimally invasive surgical procedures such as laparoscopicresections, or needle biopsies, the crosslinked, resilient foam plug canbe delivered from within a cannulated device such as a needle orcatheter, and/or through a laparoscopic device. The resilient foam plugcan be in a compressed state during delivery, and then allowed to expandonce delivered to the surgical site. The expansion of the plug cancompress the adjacent tissues to facilitate hemostasis. For thesepurposes, the expanded dimensions of the plug can provide a volume thatis at least equal to or preferably greater than the volume of the biopsyor other surgical defect, to ensure compression of surrounding tissuesby the delivered, expanded plug.

In other embodiments of the invention, methods are provided whichinclude deploying a crosslinked, resilient foam material as describedherein at a site within a bodily vessel, for example an artery or avein, so as to cause occlusion of the vessel and thereby stop the flowof fluid (e.g. blood) within the vessel. In the case of minimallyinvasive surgical procedures such as percutaneous procedures thecrosslinked, resilient foam plug can be delivered from within acannulated device such as a catheter or sheath. The resilient foam plugcan be in a compressed state during delivery, and then allowed to expandonce delivered from within the cannulated device to the desiredocclusion site. The expansion of the plug can compress the walls of thevessel to facilitate occlusion. For these purposes, the expandeddimensions of the plug can be greater than the diameter of the vessel atthe desired site of occlusion, to ensure outward compression againstsurrounding vessel walls by the delivered, expanded plug. Besidesvascular vessels, other vessels that can be occluded in accordance withthe invention include, for example, fallopian tube(s). Still further,other open tracts through patient tissue can be occluded withcrosslinked, resilient foam plugs of the invention, including forexample needle tracts (e.g. resultant of percutaneous entry to a vein orartery) and fistulas, such as anorectal fistulas, enterocutaneousfistulas, recto-vaginal fistulas, and others.

Crosslinked, resilient foam plugs can be prepared according to theinvention by a process that includes:

(a) contacting extracellular matrix material with an alkaline medium toform an expanded extracellular matrix material;

(b) washing the expanded extracellular matrix material;

(c) charging the expanded extracellular matrix material to a mold;

(d) lyophilizing the expanded extracellular matrix material in the moldto form a lyophilized extracellular matrix material foam;

(e) contacting the lyophilized extracellular matrix material foam with achemical crosslinking agent to form a crosslinked extracellular matrixmaterial foam; and

(f) drying the crosslinked extracellular matrix material foam.

In such methods, the extracellular matrix material and chemicalcrosslinked agent can, for example, be selected from among any of thosedisclosed herein. The washing can suitably be conducted with an aqueousmedium, such as saline or water. The drying can be conducted by anysuitable method, including as examples air drying at ambienttemperature, heated drying, or lyophilization. It is preferred tocontact the extracellular matrix material with the chemical crosslinkerafter the formation of the lyophilized extracellular matrix materialfoam (e.g. as opposed to incorporating the chemical crosslinker in thematerial charged to the mold), as this has been found to provide moreuniformly-shaped crosslinked plugs that resist shrinkage. Further, insuch preparative methods, the expanded extracellular matrix material canbe comminuted prior to charging to the mold. Such comminuting willprovide extracellular matrix fragments, e.g. randomly generated, thatwill be incorporated within and characterize the extracellular matrixfoam. In more preferred forms, the material is comminuted by shearingthe material with a rotating blade, e.g. in a blender. For thesepurposes, it has been discovered that when utilizing an extracellularmatrix material that is a harvested, deceullarized sheet, the sheet canbe contacted with the alkaline medium under conditions sufficient tosubstantially reduce the tensile strength of the sheet, so that thesheet material is disrupted by the rotating blade. Without sufficientreduction of tensile strength, the sheet material can tend to wraparound the rotating blade, thus frustrating the process of comminution.For example, prior to comminution by the blade or otherwise, the sheetcan be treated with the alkaline medium for a time and under conditionssufficient to reduce the tensile strength of the sheet to less thanabout 50% of its original tensile strength, more preferably to less thanabout 30% of its original tensile strength. Such methods can bepracticed, for example, with harvested sheet-form ECM materials such assubmucosa-containing sheets, e.g. obtained from small intestinal,stomach or bladder tissue, pericardial tissue, peritoneal tissue,fascia, dermal tissue, and other sheet-form ECM materials.

In additional embodiments of the invention, bioactive compositeextracellular matrix material products are used. The composite productcomprises a dried body formed with an extracellular matrix material thathas been treated with an alkaline medium under conditions effective toproduce an expanded extracellular matrix material, particles of abioactive extracellular matrix material entrapped within said driedbody, wherein the particles of bioactive extracellular matrix materialretain at least one growth factor from a source tissue for theparticulate extracellular matrix material. The composite products can beprepared by:

(a) contacting extracellular matrix material with an alkaline medium toform an expanded extracellular matrix material;

(b) washing the expanded extracellular matrix material;

(c) preparing a mixture including a liquid, the expanded extracellularmatrix material and a particulate extracellular matrix material, theparticulate extracellular matrix material retaining an amount of atleast one growth factor from a source tissue for the particulateextracellular matrix material; and

(d) drying the mixture to form a bioactive, composite extracellularmatrix material construct.

In such composite products and preparative methods, the extracellularmatrix material that is expanded, and the particulate extracellularmatrix material, can, for example, be selected from among any of thosedisclosed herein. The washing can suitably be conducted with an aqueousmedium, such as saline or water. The liquid for preparing the mixturecan be any suitable liquid, preferably biocompatible, and typically anaqueous liquid such as water or saline. The drying step can be conductedby any suitable method, including as examples air drying at ambienttemperature, heated drying, or lyophilization. Further, in suchpreparative methods, the expanded extracellular matrix material isdesirably comminuted prior to or during the formation of the mixture. Inmore preferred forms, the material is comminuted by shearing thematerial with a rotating blade, e.g. in a blender, alone or in thepresence of the bioactive particulate extracellular matrix material.Such methods can be practiced, for example, with harvested sheet-formECM materials such as submucosa-containing sheets, e.g. obtained fromsmall intestinal, stomach or bladder tissue, pericardial tissue,peritoneal tissue, fascia, dermal tissue, and other sheet-form ECMmaterials. The expanded ECM material and the bioactive particulate ECMmaterial can be from the same ECM starting material or from differentECM starting materials. The incorporation of the particulate ECMmaterial can serve not only to enhance the bioactivity of the foamproduct, but also they enhance the resiliency of the foam product. Theseaspects can be used to advantage in hemostatic, occlusion and othermedical treatments described herein.

Additional embodiments utilize composite extracellular matrix materialproducts that include an extracellular matrix sheet material and a driedmaterial adhered to the extracellular matrix sheet material, wherein thedried material is formed from an extracellular matrix material that hasbeen contacted with an alkaline medium to form an expanded extracellularmatrix material. Such composite products can be prepared by a method theincludes the steps of:

(a) contacting extracellular matrix material with an alkaline medium toform an expanded extracellular matrix material;

(b) washing the expanded extracellular matrix material;

(c) casting a flowable, wet preparation of the expanded extracellularmatrix material against an extracellular matrix sheet to form a wetcomposite; and

(d) drying the wet composite so as to form a dried composite.

In such composite sheet-material products and preparative methods, theextracellular matrix material that is expanded, and the particulateextracellular matrix material, can, for example, be selected from amongany of those disclosed herein. The washing can suitably be conductedwith an aqueous medium, such as saline or water. The liquid forpreparing the wet preparation can be any suitable liquid, preferablybiocompatible, and typically an aqueous liquid such as water or saline.The drying step can be conducted by any suitable method, including asexamples air drying at ambient temperature, heated drying, orlyophilization. Lyophilization is preferred as it forms a more porous,resilient foam material as compared to air drying or heated drying.Further, in such preparative methods, the expanded extracellular matrixmaterial in the flowable, wet preparation is desirably comminuted. Inmore preferred forms, the material is comminuted by shearing thematerial with a rotating blade, e.g. in a blender. Such methods can bepracticed, for example, with harvested sheet-form ECM materials such assubmucosa-containing sheets, e.g. obtained from small intestinal,stomach or bladder tissue, pericardial tissue, peritoneal tissue,fascia, dermal tissue, and other sheet-form ECM materials. The expandedECM material and the sheet-form ECM material can be from the same ECMstarting material or from different ECM starting materials. Theincorporation of the sheet-form ECM material can serve not only toenhance the bioactivity of the overall product, but can also provide abarrier material and/or suturable sheet attached to the dried expandedECM material (e.g. foam). Illustratively, such constructs can be used toprovide hemostasis to surgical sites or other injured tissue. In certainmodes of practice, the construct can be placed against the bleedingtissue with the dried, expanded ECM material (especially a foam) againstthe bleeding tissue. The sheet-form ECM can then provide an additionalbarrier (besides the expanded ECM material) to protect the bleedingtissue, and or can provide a suturable sheet material which can be usedto fix the construct in place, e.g. with sutures in strand or stapleform. In specific uses, such constructs can be used to apply hemostasisto surgically-treated (e.g. subject to resection) or otherwise injuredparenchymous organ tissue, such as liver or kidney tissue. In so doing,the dried, expanded ECM material is desirably pressed against theinjured parenchymous tissue, and the sheet-form ECM material canoptionally be used to fix the construct in place, as discussed above.These and other modes of practice with the composite sheet-formconstructs will be apparent to those of ordinary skill in the art fromthe descriptions herein.

Other embodiments utilize implantable medical products that comprise adried, resilient foam body formed with an extracellular matrix materialthat has been treated with an alkaline medium sufficient to form anexpanded extracellular matrix material, and a biodegradable capsulecomponent covering at least a portion of the dried resilient foam body.The dried, resilient foam body can by any such body disclosed herein,and can be received in a compressed form within the capsule component.In certain forms, the capsule component covers at least a leading end ofthe foam body, and can serve to create a more desirable delivery profilefor the product. In additional forms, the foam body can be entirelyreceived within a capsule component, preferably in a compressed state.As the capsule component degrades and weakens after implantation, thecapsule can split or otherwise break under the force of the compressedfoam body, thus releasing the foam body to expand. The expanded foambody can then serve to provide hemostasis, occlusion and/or anothertherapeutic effect at the site of implantation. The biodegradablecapsule can be made of any suitable biodegradable material, includingfor example gelatin.

Additional embodiments involve the use of implantable medical productsthat comprise a powder material and a biodegradable capsule enclosingthe powder material. Particles of the powder material comprise a driedfoam formed with an extracellular matrix material that has been treatedwith an alkaline medium sufficient to form an expanded extracellularmatrix material. Such capsular devices can be used to effectivelydeliver and retain the powdered extracellular matrix material at a siteof implantation, for example a site for hemostasis or occlusion asdescribed herein. The powder material can serve to promote hemostasis,tissue ingrowth, or another beneficial effect at the site ofimplantation. The biodegradable capsule can be made of any suitablebiodegradable material, including for example gelatin.

For the purpose of promoting a further understanding of aspects of thepresent invention, the following specific examples are provided. It willbe understood that these examples are not limiting of the presentinvention.

Example 1

This example demonstrates the process used to prepare a disinfectedsmall intestinal submucosa tissue (i.e., non-expanded SIS), which cansubsequently be used in the preparation of various medical materials anddevices. Surface and cross section micrographs of the material aredepicted in FIGS. 1B and 1D.

A ten foot section of porcine whole intestine was extracted and washedwith water. After rinsing, this section of submucosa intestinal collagensource material was treated for about two and a half hours in 0.2%peracetic acid by volume in a 5% by volume aqueous ethanol solution withagitation. Following the treatment with the peracetic acid solution, thesubmucosa layer was delaminated in a disinfected casing machine from thewhole intestine. The resultant submucosa was then rinsed four (4) timeswith sterile water. A 1 cm by 1 cm section of this material wasextracted and stained using a solution of direct red prepared by mixing10 mg direct red in 100 mL high purity water. The section of materialwas stained for approximately 5 minutes. The stained material was washedtwice with high purity water to remove any unbound stain. The stainedmaterial was placed on a glass slide and covered with a cover slip. Amicrograph was taken (Olympus microscope) at 100× magnification of thesurface of the material. A cross section of the material was thenprepared and a similar micrograph was taken. The resulting micrographwas analyzed using Spot RT software. The surface and cross sectionmicrographs are depicted in FIGS. 1B and 1D. Both the surface and crosssection micrographs show a tightly bound collagenous matrix with noexpansion.

Example 2

This example demonstrates the process used to prepare an expanded smallintestinal submucosa tissue (i.e., expanded SIS), which can subsequentlybe used in the preparation of various medical materials and devices asdescribed herein. Surface and cross section micrographs of the materialare depicted in FIGS. 1A and 1C.

A ten foot section of porcine whole intestine was extracted and washedwith water. After rinsing, this section of submucosa intestinal collagensource material was treated for about two and a half hours in 0.2%peracetic acid by volume in a 5% by volume aqueous ethanol solution withagitation. Following the treatment with peracetic acid, the submucosalayer was delaminated in a disinfected casing machine from the wholeintestine. The resultant submucosa was then rinsed four (4) times withsterile water. 300 g of this material was soaked with agitation in 1 Lof a 1M NaOH solution at 37° C. for 1 hour and 45 minutes. The materialwas removed and rinsed in a 1 L solution of high purity water for 5minutes. This rinsing step was repeated 8 additional times. A 1 cm by 1cm section of this material was extracted and stained using a solutionof direct red prepared by mixing 10 mg direct red in 100 mL high puritywater. The section of material was stained for approximately 5 minutes.The stained material was washed twice with high purity water to removeany unbound stain. The stained material was placed on a glass slide andcovered with a cover slip. A micrograph was taken (Olympus microscope)at 100× magnification of the surface of the material. A cross section ofthe material was then prepared and a similar micrograph was taken. Theresulting micrograph was analyzed using Spot RT software. The surfaceand cross section micrographs are depicted in FIGS. 1A and 1C. Both thesurface and cross section micrographs show disruption of the tightlybound collagenous matrix and an expansion of the material.

As can be observed in FIGS. 1A-1D, both the surface view and thecross-section view of the non-expanded SIS show a tightly boundcollagenous matrix whereby collagen content is substantially uniformthroughout. Conversely, the surface view and cross-section view of theexpanded SIS show a denatured collagenous network and an expansion ofthe material.

Example 3

This Example was performed to identify additives that can be included inan expanded remodelable collagenous material for purposes of promotingrapid re-expansion of the material after implantation into a patient.

An expanded remodelable material was prepared generally as described inExample 2. Briefly, a ten foot section of porcine whole intestine wasextracted and washed with water. After rinsing, this section ofsubmucosa intestinal collagen source material was treated for about twoand a half hours in 0.2% peracetic acid by volume in a 5% by volumeaqueous ethanol solution with agitation. Following the treatment withperacetic acid, the submucosa layer was delaminated in a disinfectedcasing machine from the whole intestine. The resultant submucosa wasthen rinsed four (4) times with sterile water. 300 g of this materialwas soaked with agitation in 1 L of a 3M NaOH solution at 37° C. for 2hours. The material was removed and rinsed in a 1 L solution of highpurity water for 15 minutes. After 15 minutes, 1 L of 0.2M acetic acidwas added with agitation. After 15 minutes of agitation, the materialwas rinsed with 1 L of high purity water with shaking for 5 minutes.This rinsing step was repeated four (4) times for a total of five (5)rinses.

The rinsed material was mechanically agitated using the pulse setting ofa blender to the extent that the blended material could be transferredusing a disposable 25 mL pipette. Samples of the blended material werecombined with a handheld blender with the various additives asidentified in Table 1. The samples were then cast into cylindricalmolds, frozen at −80° C. for 5 hours, and lyophilized for 24 hours toyield 14 mm diameter cylindrical constructs ranging in length from about15 mm to about 19 mm.

TABLE 1 Additive Category Screened Additives Salts Sodium chlorideSodium acetate Sodium bicarbonate Sodium citrate Calcium carbonatePotassium acetate Potassium phosphate Hydrogels and Water AlginateSwelling Polymers Polyhydroxyethyl methacralate Polyvinyl alcoholPolyethylene glycol Carboxymethyl cellulose Polyvinyl pyrrolidoneProteins Gelatin SIS particulate Acids and Bases Acetic acid Ascorbicacid Monosaccharides and Dextran Polysaccharides Glucose FructoseSuperabsorbing Polymers Polyacrylic acid and Gelling AgentsPolygalacturonic acid Other Additives Guanidine HCI Urea

At the time of testing, the initial sample diameter was recorded. Allcylindrical samples were then compressed by hand to between 2.7 mm and6.7 mm, and the final diameter of the compressed material was recorded.Approximately 20 mL of high purity water at room temperature wastransferred into a weight boat. The compressed material was placed onthe surface of the high purity water and submerged using forceps toexpose all surfaces of the material to the high purity water. A digitaltimer was started at the time the sample was submerged. Visualassessment of the material was continuously conducted until the samplereturned to the initial sample diameter as assessed through visualinspection. When the sample returned to the initial sample diameter, thetimer was stopped and the expansion time recorded. Visual assessment wasdiscontinued after 15 minutes for samples that did not return to theinitial sample diameter in the time allotted. The results are summarizedin Tables 2-8.

TABLE 2 Initial Compressed Expansion % Dry Weight Diameter Diameter TimeAdditive of Dry Plug (mm) (mm) (min:sec) Sodium 2.5 12 4.0  1:41chloride 7.5 13 4.0 >15:00* 14 3.7 >15:00* Sodium acetate 1.25 13 3.7 6:30 13 3.7  6:00 2.5 13 4.7  0:45 12 4.3  0:45 5.0 13 4.7  1:30 14 5.0 2:00 Sodium 2.5 13 4.0  2:00 bicarbonate 13 4.3  1:15 5.0 13 6.7  3:0013 5.0  1:20 Sodium citrate 2.5 14 5.3  8:00 14 5.0  8:00 5.0 14 5.012:00 14 4.7 12:00 Calcium 2.5 14 4.7 >15:00* carbonate 14 4.3 >15:00*5.0 14 5.3  8:00 14 5.0  8:00 12.5 14 4.7 >15:00* 14 4.7 >15:00*Potassium 2.5 13 5.3 >15:00* acetate 13 4.7 >15:00* 5.0 14 4.3 >15:00*14 4.3 >15:00* Potassium 2.5 10 3.0 13:00 phosphate 10 3.0 13:00 5.0 133.0 14:00 12 3.3 11:00 *Indicates control sample behaved atypically,suggesting the expansion time may not be representative of the additivetested.

TABLE 3 Initial Expansion % Dry Weight Diameter Diameter Time Additiveof Dry Plug (mm) (mm) (min:sec) Alginate 2.5 13 3.0 >15:00 Polyhydroxyethyl 2.5 13 3.0 8:50 methacralate 13 2.7 8:58 Polyvinylalcohol 2.5 14 3.0 5:48 Polyethylene 7.5 14 2.3 >15:00*  glycol (MW 400)14 2.3 >15:00*  Polyethylene 2.5 13 3.0 3:22 glycol (MW 6000)Carboxymethyl 2.5 13 3.7 7:03 cellulose Polyvinyl 2.5 14 3.3 5:25pyrrolidone *Indicates control sample behaved atypically, suggesting theexpansion time may not be representative of the additive tested.

TABLE 4 Initial Expansion % Dry Weight Diameter Diameter Time Additiveof Dry Plug (mm) (mm) (min:sec) Gelatin (100 2.5 13 3.0 >15:00  bloom)45-90 μm SIS 5.0 14 4.7 2:38 particulate 13 4.7 2:35 10.0 13 5.0 1:32 134.7 1:20 20.0 14 6.3 0:37 14 6.0 0:52 90-150 μm SIS 5.0 14 3.7 2:30particulate 13 3.7 2:00 10.0 13 4.7 2:30 14 5.0 3:00 20.0 13 5.3 1:30 136.3 1:42 150-200 μm SIS 5.0 14 4.0 2:45 particulate 14 4.3 2:50 10.0 144.7 2:30 13 4.3 2:25 20.0 13 5.7 1:55 13 5.0 2:35

TABLE 5 Initial Expansion % Dry Weight Diameter Diameter Time Additiveof Dry Plug (mm) (mm) (min:sec) Ascorbic acid 2.5 14 3.0 >15:00* 143.0 >15:00* 5.0 14 3.0 >15:00* 14 3.3 >15:00* *Indicates control samplebehaved atypically, suggesting the expansion time may not berepresentative of the additive tested.

TABLE 6 Initial Expansion % Dry Weight Diameter Diameter Time Additiveof Dry Plug (mm) (mm) (min:sec) Polyacrylic acid 2.5 13 3.3 8:24 13 3.08:07 Polygalacturonic 2.5 13 3.0 4:00 acid 13 3.0 4:35

TABLE 7 Initial Expansion % Dry Weight Diameter Diameter Time Additiveof Dry Plug (mm) (mm) (min:sec) Dextran 2.5 13 3.0  5:15 13 3.3  4:16Glucose 2.5 14 3.7 >15:00* 14 3.7 >15:00* 5.0 14 3.7 >15:00* 143.0 >15:00* Fructose 2.5 14 3.7 >15:00* 14 4.0 >15:00* 5.0 143.3 >15:00* 14 3.7 >15:00* *Indicates control sample behaved atypically,suggesting the expansion time may not be representative of the additivetested.

TABLE 8 Initial Compressed Expansion % Dry Weight Diameter Diameter TimeAdditive of Dry Plug (mm) (mm) (min:sec) Guanidine HCI 2.5 14 3.0 4:1614 2.7 4:50 Urea 5.0 14 3.0 >15:00  14 3.3 >15:00 

Based on these results, preferred additives include sodium chloride,sodium acetate, sodium bicarbonate, polyethylene glycol (MW 6000), andsmall intestinal submucosa particulate

Example 4

This Example was performed to measure the angiogenic activity of variousforms of an expanded remodelable collagenous material as describedherein.

An expanded remodelable material was prepared generally as described inExample 3. Briefly, a ten foot section of porcine whole intestine wasextracted and washed with water. After rinsing, this section ofsubmucosa intestinal collagen source material was treated for about twoand a half hours in 0.2% peracetic acid by volume in a 5% by volumeaqueous ethanol solution with agitation. Following the treatment withperacetic acid, the submucosa layer was delaminated in a disinfectedcasing machine from the whole intestine. The resultant submucosa wasthen rinsed four (4) times with sterile water. 300 g of this materialwas soaked with agitation in 1 L of a 3M NaOH solution at 37° C. for 2hours. The material was removed and rinsed in a 1 L solution of highpurity water for 15 minutes. After 15 minutes, 1 L of 0.2M acetic acidwas added with agitation. After 15 minutes of agitation, the materialwas rinsed with 1 L of high purity water with shaking for 5 minutes.This rinsing step was repeated four (4) times for a total of five (5)rinses. Three different forms of expanded remodelable collagenousmaterial were prepared from this material: (1) blended expandedremodelable collagenous material, (2) expanded remodelable collagenousmaterial in conjunction with a submucosa particulate (1:10), and (3)4-layered lyophilized sheet form expanded remodelable collagenousmaterial.

These materials from groups (1) and (2) were cast into a thick film ofapproximately 1 mm in thickness, frozen at −80° C. for 5 hours andlyophilized for 24 hours. Ten 15 mm discs were cut from each group usinga disc punch to form test samples. Nylon filters with 0.22 μm pores weresewn on to the top and bottom of each disc. Low temperature ethyleneoxide sterilization was used for each sample. Samples were implantedsubcutaneously into the dorsal flanks of mice. After anesthesia usingKetamine (87 mg/kg) and Xylazine (13 mg/kg), a small incision was madeon the posterior neck of the mouse, and a dorsal subcutaneous cavity wascreated using blunt dissection with hemostats. This was followed bysample placement and closure of the incision with 4 interrupted stitchesof 5-0 suture. Six mice per group underwent disc implantation. Theimplant remained in the mice for a period of 3 weeks followed by probingfor capillary formation.

Mice were sacrificed using a double dose of anesthesia to ensure intactflow in vasculature. While the heart was still beating, the chest cavitywas exposed, vena cava severed, and 10 mL of heparized saline injectedinto the left ventricle using a 23 ga butterfly infusion set toexsanguinate the mouse. After transferring syringes (while maintaininginfusion needle in left ventricle), 4 mL of a fluorescent microsphere(yellow-green, 0.1 μm diameter, Molecular Probes, F-8803) suspension(1:20 dilution of stock suspension) was injected through the leftventricle resulting in perfusion of the entire vasculature. Care wastaken to ensure no bubbles were introduced during the injections, asbubbles will cause micro-emboli obstructing consistent perfusion.Samples were collected with gentle dissection and gross removal of thefibrous capsule. A positive control of hind limb muscle was alsocollected at this point to confirm proper perfusion. Collected samplesand controls were placed on ice in a closed container to maintain tissueintegrity (mainly moistness). Microvasculature was imaged using aconfocal microscope (Biorad), λ_(ex)=488 nm & λ_(em)=530 nm, along theedge of the samples in the area of greatest vascular infiltration.Further, vasculature of the positive controls, hind limb muscle, wasimaged to confirm good perfusion.

In addition to the fluorescence microangiography described above,samples were collected, placed in histology cassettes, and submerged in10% buffered formalin (Fisher). Histological sectioning and stainingwith hematoxilin and eosin were performed by Portland Tissue Processing.Images of H&E stained sections of the disc edge for each sample weretaken using a microscope (Olympus) with a 10× objective.

Each of the samples from all three test groups showed some angiogenicactivity when fluorescence microangiography was performed. Similarly,the histology analysis confirmed that all three sample groups had somevascular and cellular ingrowth.

This Example demonstrates that various forms of an expanded remodelablecollagenous material each exhibit angiogenic activity in vivo.

Example 5

This Example was performed to investigate the angiogenic activity of acrosslinked, expanded remodelable collagenous material as describedherein.

An expanded remodelable material was prepared generally as described inExample 3. Briefly, a ten foot section of porcine whole intestine wasextracted and washed with water. After rinsing, this section ofsubmucosa intestinal collagen source material was treated for about twoand a half hours in 0.2% peracetic acid by volume in a 5% by volumeaqueous ethanol solution with agitation. Following the treatment withperacetic acid, the submucosa layer was delaminated in a disinfectedcasing machine from the whole intestine. The resultant submucosa wasthen rinsed four (4) times with sterile water. 300 g of this materialwas soaked with agitation in 1 L of a 3M NaOH solution at 37° C. for 2hours. The material was removed and rinsed in a 1 L solution of highpurity water for 15 minutes. After 15 minutes, 1 L of 0.2M acetic acidwas added with agitation. After 15 minutes of agitation, the materialwas rinsed with 1 L of high purity water with shaking for 5 minutes.This rinsing step was repeated four (4) times for a total of five (5)rinses. Approximately 250 mL of the expanded remodelable collagenousmaterial was placed into a blender along with 250 mL of high puritywater. This mixture was pulsed 10 times for 1 second each pulse followedby a 45 second blend. The resulting material was cast into a 5×10 cmmold having a thickness of approximately 1 mm. This mold was placed in afreezer at −80° C. for 5 hours followed by lyophilization for 24 hours.15 mm disc samples were cut from the resulting blended sheet.

To form the crosslinked samples, the samples formed above were combinedwith 200 mL of 50 mM EDC crosslinking solution in a shallow glass dish.The disc with samples were submerged under solution and placed onto arotating shaker for 24 hours at room temperature. Each sample was thenrinsed with 200 mL of high purity water squeezing five (5) times. Thisstep was repeated four (4) times for a total of five (5) rinses. Therinsed material was then lyophilized for approximately 8 hours.

Each of the samples showed some angiogenic activity when fluorescencemicroangiography was performed. Similarly, the histology analysisconfirmed that all three sample groups had some vascular and cellularingrowth. Indeed, the crosslinked material had robust angiogenesis(1442+108 μm) and was still present in plug form. The plug expanded atexplant indicating that the crosslinked material was substantive and didnot collapse after implantation. Moreover, there were no signs ofsystemic or local toxicity and no evidence of increased localinflammation in these samples.

This Example further demonstrates that a crosslinked form of an expandedremodelable collagenous material can exhibit angiogenic activity invivo.

Example 6

This Example was performed to determine the FGF-2 content of an expandedremodelable collagenous material as described herein.

An expanded remodelable material was prepared generally as described inExample 3. Briefly, a ten foot section of porcine whole intestine wasextracted and washed with water. After rinsing, this section ofsubmucosa intestinal collagen source material was treated for about twoand a half hours in 0.2% peracetic acid by volume in a 5% by volumeaqueous ethanol solution with agitation. Following the treatment withperacetic acid, the submucosa layer was delaminated in a disinfectedcasing machine from the whole intestine. The resultant submucosa wasthen rinsed four (4) times with sterile water. 300 g of this materialwas soaked with agitation in 1 L of a 3M NaOH solution at 37° C. for 2hours. The material was removed and rinsed in a 1 L solution of highpurity water for 15 minutes. After 15 minutes, 1 L of 0.2M acetic acidwas added with agitation. After 15 minutes of agitation, the materialwas rinsed with 1 L of high purity water with shaking for 5 minutes.This rinsing step was repeated four (4) times for a total of five (5)rinses.

Two lots of material described above were prepared with one lot used pergroup. One lot of material was made into single-layer lyophilizedsheets, and the other material was mixed with small intestinal submucosaparticulate (˜150 μm) and made into single-layer lyophilized sheets.Three (3) samples were cut (2 cm×2 cm) from each lot resulting in three(3) samples per group. Each sample was weighed and its weight wasrecorded. Individual samples were placed in 1.5 mL eppendorf tubes and400 μl of sterile phosphate buffered saline (PBS) was added to eachtube. Tubes with samples were centrifuged at 12000 g for 5 minutes at 4°C. The resulting supernatant was diluted to 1:1 with 1×PBS. Samples wereassayed in duplicate for FGF-2 content using R&D Systems FGF-2 ELISAkits per manufacturer's instructions.

The resulting content of FGF-2 was calculated by dividing the FGF-2content by the weights of the samples. The means measured FGF-2 contentin the sheet form expanded remodelable collagenous material was 0 pg/g.The mean measured FGF-2 content in expanded remodelable collagenousmaterial including a submucosa particulate was 4500 pg/g+1600 pg/g.

This Example demonstrates that an expanded remodelable collagenousmaterial in sheet form, prepared and tested as described in thisexample, contains no detectable levels of FGF-2, and that FGF-2 can beprovided back to an expanded remodelable collagenous material by virtueof the inclusion of a submucosa particulate into the material.

The use of the terms “a” and “an” and “the” and similar references inthe context of describing the invention (especially in the context ofthe following claims) are to be construed to cover both the singular andthe plural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations of those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than as specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context. In addition, all publications cited herein areindicative of the abilities of those of ordinary skill in the art andare hereby incorporated by reference in their entirety as ifindividually incorporated by reference and fully set forth.

1. A method for tissue biopsy with applied hemostasis, comprising:advancing a biopsy device into tissue of a patient; cutting a biopsysample from a location in said tissue; removing the biopsy sample fromthe patient; and implanting a hemostatic biopsy plug at said location insaid tissue, said biopsy plug comprising a resilient foam body formedwith an extracellular matrix material that has been treated with analkaline medium sufficient to form an expanded extracellular matrixmaterial.
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. The method ofclaim 1, wherein said extracellular matrix material is a decellularizedtissue layer.
 6. The method of claim 5, wherein said decellularizedtissue layer comprises submucosa.
 7. (canceled)
 8. (canceled)
 9. Themethod of claim 1, wherein said expanded extracellular matrix materialis dried by lyophilization.
 10. The method of claim 1, wherein saidtissue of said patient is kidney or liver tissue.
 11. A hemostatictissue biopsy plug product, comprising: a resilient, hemostaticextracellular matrix foam body sized for receipt at a tissue biopsysite, said foam plug formed with an extracellular matrix material thathas been treated with an alkaline medium sufficient to form an expandedextracellular matrix material, said foam plug compressible to acompressed condition having a greatest cross-sectional dimension notexceeding 5 mm and expandable to an expanded condition having a greatestcross-sectional dimension of at least 10 mm.
 12. The plug product ofclaim 11, wherein said expanded extracellular matrix material iscomprised of a dry lyophilized material.
 13. The plug product of claim11, wherein said construct further comprises at least one biologicallyactive agent.
 14. The plug product of claim 13, wherein said at leastone biologically active agent includes one or more of a growth factor,glycoprotein, glycosaminoglycan, or proteoglycan.
 15. A product forapplying hemostasis to a biopsy site, comprising: a cannulated devicehaving a lumen, said cannulated device advanceable to a tissue biopsysite; and a hemostatic tissue biopsy plug according to claim 12 receivedin said lumen.
 16. A method for providing hemostasis at a surgical site,comprising: surgically treating tissue at a site in a patient in such amanner as to cause bleeding at the site; and applying a hemostaticextracellular matrix foam to the site so as to cause hemostasis, saidfoam formed with an extracellular matrix material that has been treatedwith an alkaline medium sufficient to form an expanded extracellularmatrix material.
 17. (canceled)
 18. (canceled)
 19. The method of claim16, wherein said extracellular matrix material is a decellularizedtissue layer.
 20. The method of claim 16, wherein said expandedextracellular matrix material is dried by lyophilization.
 21. The methodof claim 16, wherein said surgically treated tissue is kidney or livertissue.
 22. A method for surgical removal of parenchymal tissue in apatient with applied hemostasis, comprising: performing a partialnephrectomy or hepatectomy in a patient so as to cause bleeding in akidney or liver, respectively, of the patient; applying a hemostaticextracellular matrix foam to the kidney or liver so as to causehemostasis, said foam formed with an extracellular matrix material thathas been treated with an alkaline medium sufficient to form an expandedextracellular matrix material.
 23. The method of claim 22, wherein saidextracellular matrix material is a decellularized tissue layer.
 24. Themethod of claim 22, wherein said expanded extracellular matrix materialis dried by lyophilization.
 25. A method for preparing a compressiblemedical foam product, comprising: contacting an extracellular matrixmaterial with an alkaline medium to form an expanded extracellularmatrix material; washing the expanded extracellular matrix material;charging the expanded extracellular matrix material to a mold; andlyophilizing the expanded extracellular matrix material in the mold toform a lyophilized extracellular matrix material foam; contacting thelyophilized extracellular matrix material foam with a chemicalcrosslinking agent to form a crosslinked extracellular matrix foam; anddrying the crosslinked extracellular matrix material foam to form thecompressible medical foam product.
 26. (canceled)
 27. (canceled) 28.(canceled)
 29. The method of claim 25, wherein said extracellular matrixmaterial is a decellularized tissue layer.
 30. The method of claim 29,wherein said decellularized tissue layer comprises submucosa. 31.(canceled)
 32. (canceled)
 33. The method of claim 25, wherein saidexpanded extracellular matrix material is dried by lyophilization. 34.The method of claim 25, wherein said tissue of a patient is kidney orliver tissue.
 35. A compressible medical foam product, comprising: adried, compressible foam body, said foam body formed with anextracellular matrix material that has been treated with an alkalinemedium sufficient to form an expanded extracellular matrix material,wherein said foam body has introduced chemical crosslinks sufficient toincrease the resiliency of the foam body.
 36. (canceled)
 37. Thecompressible medical foam product of claim 35, wherein saidextracellular matrix material is a decellularized tissue layer.
 38. Thecompressible medical foam product of claim 37, wherein saiddecellularized tissue layer comprises submucosa.
 39. (canceled) 40.(canceled)
 41. The compressible medical foam product of claim 35,wherein said expanded extracellular matrix material is comprised of adry lyophilized material.
 42. The compressible medical foam product ofclaim 35, wherein said construct further comprises at least onebiologically active agent.
 43. The compressible medical foam product ofclaim 35, wherein said at least one biologically active agent includesone or more of a growth factor, glycoprotein, glycosaminoglycan, orproteoglycan.